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O R I GI N A L A R T IC L E
Received: 13 May 2005 / Accepted: 9 September 2005 / Published online: 28 October 2005
SBIC 2005
Abstract Ever since it was proposed that reactive oxygen antioxidative activity in biological systems. In conclu-
species (ROS) are involved in the pathogeneses of vari- sion, Cu2(asp)4 is a potent antioxidative agent that may
ous diseases, superoxide dismutase (SOD)-mimetic be useful for future treatment of diseases resulting from
complexes have been intensively studied. We prepared ROS.
copper(II) aspirinate [Cu2(asp)4] from Cu(II) and aspi-
rin, which has been in use for many years as an anti- Keywords Copper aspirinate Æ Copper salicylate Æ
pyretic, an analgesic, and an anti-inflammatory agent. Copper acetate Æ Copper sulfate Æ HaCaT cell Æ
However, Cu2(asp)4 has been found to have additional Fibroblasts Æ Reactive oxygen species Æ Skin
activities, including anti-inflammatory, antiulcer, anti-
ischemic/reperfusion agent, anticancer, antimutagenic,
and antimicrobial activities. The activity of copper Introduction
salicylate [Cu(sal)2] was also compared with that of
Cu2(asp)4. The structure of the Cu2(asp)4 was deter- It has been proposed that reactive oxygen species (ROS)
mined using X-ray structure analysis. Its SOD-mimetic are involved in the pathogeneses of various diseases,
activity was determined usingcytochrome c, electron such as lifestyle-related diseases including hypertension
spin resonance (ESR) spectroscopy, and ESR spin trap [1] and photoaging due to exposure to ultraviolet radi-
methods. The activity of Cu2(asp)4 was slightly greater ation. Cu-dependent and Zn-modulated cytosolic and
than CuSO4 and copper acetate [Cu(ace)2] and slightly extracellular superoxide dismutases (Cu2Zn2SOD),
less than that of Cu(sal)2. The in vitro antioxidant which are present in a wide range of animals (including
activity, evaluated in human epithelial or transformed man), catalyze the dismutation reaction of superoxide
neoplastic keratinocyte cells, HaCaT, and normal der- (ÆO2 ) to yield hydrogen peroxide (H2O2) and triplet state
mal fibroblasts in terms of cell survival following ultra- oxygen (3O2) [2]. SOD activity in mammalian cells,
violet B (UVB) irradiation, was significantly increased in including those of man, tends to decrease with age;
the presence of Cu2(asp)4, Cu(sal)2, and CuSO4. Fur- hence, the intake of SOD-enhancing compounds capable
ther, ROS generation following UVA irradiation in the of facilitating de novo synthesis of Cu2Zn2SOD is rec-
skin of hairless mice following oral treatment with ommended [3]. Since SOD is labile to gastric and intes-
Cu2(asp)4 for three consecutive days was significantly tinal proteases, which cause digestion of this enzyme,
suppressed compared to the vehicle- or Cu(ace)2-treated oral intake of small-molecular-mass SOD-mimetic
mice. On the basis of these results, Cu2(asp)4 was ob- complexes with similar Cu2Zn2SOD active site struc-
served to be a potent antioxidative compound possessing tures was originally proposed by Sorenson [4] and then
intensively developed by many researchers [5–11].
T. Fujimori Æ S. Yamada Æ H. Yasui Æ H. Sakurai (&) During our study of the development of SOD--
Department of Analytical and Bioinorganic Chemistry, mimetic metalloelement complexes [12–14], we planned
Kyoto Pharmaceutical University, 5 Nacauchi-cho, the preparation of Cu(II) complexes with pharma-
Misasagi, Yamashina-ku, Kyoto 607-8414, Japan cologically active ligands. It is well known that Cu(II)
E-mail: sakurai@mb.kyoto-phu.ac.jp
Tel.: +81-75-5954629 has superoxide scavenging activity and that aspirin has
Fax: +81-75-5954753 been used for many years as an antipyretic, an analgesic,
and an anti-inflammatory agent; in recent years it has
Y. In Æ T. Ishida also been used as a platelet anti-aggregating agent [15,
Department of Physical Chemistry,
Osaka University of Pharmaceutical Sciences,
16], an antitumor agent [17], and as a protective agent
4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan against UV exposure [18, 19]. Subsequently, we prepared
832
ESR parameters
Determination of physicochemical properties of the 550 nm using a Ubest-55 UV–VIS spectrometer (Jasco,
complexes Tokyo, Japan) in the presence of different amounts of
Cu(II) complexes dissolved in DMSO and CuSO4 in
The IR spectra were obtained using the KBr disc method deionized water. The increase (DA550) in the absorbance
with a FTIR-8100A spectrophotometer (Shimazu, Ja- was monitored 45 s after the initiation of the reaction
pan). The ESR spectra were obtained with an X-band for 90 s. The SSA of a compound was expressed as an
ESR spectrometer RE-1X (Joel Ltd., Tokyo, Japan). The IC50 value, which defines 50% of the inhibitory con-
visible absorption spectra were obtained using a DMSO centration of a compound in the reaction [26].
solution and a KBr disc with an Agilent spectropho-
tometer (Yokogawa Analytical Systems, Tokyo, Japan) Direct ESR spectroscopic method for the measurement of
and MCPD-1000 spectrophotometer (Otsuka Electron- SSA
ics, Osaka, Japan), respectively. Magnetic susceptibilities
were obtained with a magnetic susceptibility balance Both 3.17 mg (0.045 mmol) of KO2 and 27.8 mg
(MK I, Sherwood Scientific, Cambridge, England). (0.077 mmol) of DB18C6 were combined in a test tube
with 5 ml of a mixture of toluene and DMSO (3:2 v/v).
X-ray structure determination of Cu2(asp)4 The resultant solution was purged with argon gas for
60 s and sealed with a septum and then sonicated for
A single crystal with dimensions 0.4·0.2·0.20 mm3 was 2 min. The supernatant was used for ESR measure-
used for X-ray crystallography. The X-ray data were ments. One hundred microliters of different concentra-
collected with a Bruker (Karlsruhe, Germany) SMART tions of the test compounds in DMF (ranging from 0 to
APEX CCD camera using graphite-monochromated 10 lM) was added to 200 lL of the KO2 and DB18C6
MoK (k=0.71073 Å) at 120 K. The structure was toluene/DMSO solution in a /=5 mm quartz ESR cell.
solved by a direct method using the SHELXS97 pro- After mixing, the solution was frozen at liquid nitrogen
gram [23]. Atomic scattering factors were taken from temperature and the resultant ESR spectrum was re-
international tables for X-ray crystallography [24]. The corded using an X-band ESR spectrometer RE-1X
positional parameters of non-H atoms were refined by a (JOEL) [27].
full matrix least squares method with anisotropic ther-
mal parameters using the program SHELXL97 [25].
ESR spin trap method using DMPO
JES-RFR30 (JOEL). The ratio of the signal intensity (S) 480 lmol Cu(sal)2 kg1, 480 lmol Cu(ace)2 kg1,
appearing at the lowest magnetic field due to the 960 lmol aspirin kg1 or 960 lmol salicylic acid kg1
DMPO–OO(H) adduct to that of the third signal were administered orally for three consecutive days to
intensity (M) due to Mn(II) was termed the S/M ratio. six-week-old male hairless mice (HR-1). These mice
The SSA of the Cu(II) compound added in DMSO or were anesthetized by intraperitoneal injection of 50 mg
water ranging in concentration from 0 to 20 lM was pentobarbital kg1 body mass and placed on a heating
determined based upon the IC50 value, which defines the pad for 30 min after last treatment. The skin surfaces
50% inhibitory concentration of the compound showing of these mice were carefully cleaned with aqueous 50%
the 50% S/M ratio to vehicle control [28]. ethanol and each mouse was covered with a black cloth
in which two holes were cut. The holes on the right
Singlet oxygen Singlet oxygen formed in a hemato- sides of the mice were irradiated with 18 J cm2 UVA,
porphyrin (0.25 mM phosphate buffer, pH 7.4) irradi- while the left side was unexposed; subsequently,
ated with UVA light (through a UVA filter at a dose of 200 lM CLA (10 lL) was applied to the measurement
800 mW cm2 using a Superure-203S, San-Ei Electric areas of the skin of the mouse. Chemiluminescence
MFG, Japan) was trapped with 200 mM TMPD and its emitted from a single given area (78.5 mm2) in the skin
ESR spectrum was recorded using an X-band ESR below the hole was measured for 30 m with a super-
spectrometer JES-RFR30 (JOEL) in a manner similar to sensitive chemiluminescence-measuring apparatus, a
that employed for superoxide trapping, using 0–150 lM NightOWL (luminograph LB 981, Wallack Berthold,
Cu(II) compounds added in DMSO or water [28]. Germany), which provides chemiluminescent (CL)
intensity (photons/pixel/second) as well as CL imaging.
The area under the curve (AUC) of measured CL
intensities vs time was evaluated (mega photons/pixel)
Cell cultures and UVB irradiation [32, 33].
In vivo chemiluminescent measurement All experimental results are expressed as the mean ±
in hairless mice standard deviation (SD) of triplicate determinations.
Statistical analysis was performed by analysis of vari-
Compounds were suspended in 5% acacia–deionized ance (ANOVA) at a 1% or 5% significance level of
water solution and doses of 240 lmol Cu2(asp)4 kg1, difference.
835
Fig. 1a–d ESR spectra of Cu2(asp)4 at room temperature (a), at 77 K (b); Cu(sal)2 at room temperature (c); and Cu(sal)2 at 77 K (d),
dissolved in DMSO
on Cu concentrations; however, doses above 15 lM of hairless mouse skin treated with Cu2(asp)4 or Cu(sal)2
caused cell toxicity (Fig. 5). The order of cell survival orally for three consecutive days was significantly sup-
was: Cu(sal)2 > Cu2(asp)4 > CuSO4. pressed as compared with vehicle-treated control and
Cu(ace)2-, aspirin-, or salicylic acid-treated mice (Fig. 6).
It is noteworthy that the administration of Cu(ace)2,
Suppression of reactive oxygen species generated with which was less toxic than CuSO4, exhibited no inhibition
UVA irradiation of hairless mouse skin with Cu2(asp)4 of ROS generation in the skin of each mouse. These
results show that Cu2(asp)4 had a greater in vivo ROS
The UVA-induced generation of ROS (evidenced by suppressing effect than the other copper(II) compounds
chemiluminescence intensity) following UVA-irradiation (Table 5).
Complex Cu–Cu Cu–O Cu–O (axial) O–C Cu–basal Cu–O–C O–C–O () Reference
(Å) (basal) (Å) (Å) (carboxylate) plane (Å) ()
(Å)
The distribution of Cu in ICR mice treated with Cu(II) The X-ray structure analysis of Cu2(asp)4(DMSO)2
compounds and ligands orally for three consecutive days indicates that its crystal structure is binuclear with a
was determined by AAS. The blood Cu concentrations crystallographic center of symmetry (Fig. 2). Four
30 and 90 min after the last administration of the com- aspirin molecules are bonded to two Cu(II) atoms
pounds on day 3 and the skin Cu concentrations 90 min through four carboxylate bridges to form a Cu2(CO2)4
after the last administration are shown in Table 6. The unit. Each Cu(II) atom forms a hemi-octahedral coor-
Cu concentration of the Cu(ace)2 group was increased dinated moiety with the equatorial positions occupied by
30 min after the last administration, although the Cu four carboxylate oxygens of two aspirin molecules, and
concentration of the Cu(ace)2 group was normal 90 min with the axial positions occupied by the oxygen atom of
after the administration. On the other hand, the Cu DMSO. The crystal structure of Cu2(asp)4 [21] and the
concentrations of the Cu2(asp)4 and Cu(sal)2 groups DMF solvate, Cu2(asp)4(DMF)2 [36], have been previ-
were slightly increased 90 min after administration. The ously reported [21]. In the present study, we analyzed the
Cu concentration in the skin treated with Cu2(asp)4 was crystal structure of the DMSO solvate (Table 2). The
slightly increased. axial coordination positions of the Cu(II) atoms in
Cu2(asp)4 are occupied by neighboring acetyl oxygen DMSO or the neighboring acetyl group of Cu2(asp)4,
atoms of Cu2(asp)4, whereas these positions are occu- and the Cu–Cu bond lengths are: 1.963(3), 2.131(3), and
pied by oxygen atoms of DMSO in the Cu2(asp)4(DM- 2.632(1) Å, respectively, for Cu2(asp)4(DMSO)2;
SO)2 crystal. The coordination bonds of both complexes 1.963(4), 2.241(8), and 2.617(3) Å, respectively, for
indicate that the average Cu–O bond length for the four Cu2(asp)4 with the axially bonded acetyl group of a
equatorially coordinated oxygen atoms of these carb- neighboring molecule of Cu2(asp)4 [21]; and 1.953(12),
oxylates, the Cu–O axially coordinated oxygen atom of (and 1.971(11) Å for the second molecule of DMF),
2.154(12) Å, and 2.615(4) Å, respectively, for the
Table 5 Suppression of reactive oxygen species generation with
UVA irradiation of HR-1 hairless mouse skin after oral treatment Cu2(asp)4(DMF)2 solvate [36] (Table 3). The coordina-
for three consecutive days tion bond length at the axial position is significantly
shorter in Cu2(asp)4(DMSO)2 than in Cu2(asp)4(DMF)2,
Treatment % Suppression while the Cu–Cu bond length is longer in
vs. control
Cu2(asp)4(DMSO)2 than the others, which may be due
Cu2(asp)4 44 to the greater nucleophilicity of DMSO. This difference
Cu(sal)2 35 is also reflected in the magnetic susceptibilities of these
Aspirin 17 compounds (Table 1).
Salicylic acid 19 The ESR spectra showed that Cu2(asp)4 exists only in
Cu(ace)2 0
the mononuclear state, Cu(asp)2, in this polar solvent, as
840
Table 6 Cu concentration in the blood and skin of ICR mice treated with Cu compounds or ligands
indicated previously in other Cu(II) complexes [38]. Encouraged by the enhancement of cell survival due
Since the Cu atoms account for the antioxidative activ- to the presence of Cu(II) compounds, we examined the
ity, the present results demonstrate that coordination ROS suppressive effects of Cu(II) complexes in the skin
structure of the metalloelement complex is important for of hairless mice following oral treatment. Cu2(asp)4
biological activity [10]. significantly suppressed ROS generation in the skin of
Based upon results obtained for three different eval- UVA-irradiated mice more effectively than Cu(sal)2 or
uation systems for ÆO 2 scavenging activity (Table 4), the control aspirin- or salicylic acid-treated mice, where Cu
activities of these Cu(II) compounds were found to be complexes of salicylic acid or aspirin may have formed
essentially equivalent, which is attributed to the reac- in vivo [8] (Table 5; Fig. 6). Previously, we proposed
tivities of mononuclear complexes in all cases. Although that both superoxide and singlet oxygen (1O2) are
these activities are approximately only 1.6·103 times formed during the exposure of the skin of live animals to
the reactivity of Cu2Zn2SOD, which has little or no UVA[31]. In the present study, it was observed that
activity when studied in test systems in vivo, small Cu2(asp)4 did not exhibit 1O2 scavenging activity but did
molecular mass complexes are effective in systems in show ÆO 2 scavenging activity (Table 4); therefore, the
vivo [8, 9] as well as in the xanthine/xanthine oxidase/ suppression of ROS generated in the UVA-exposed skin
cytochrome c and ESR in vitro systems. These results by the Cu2(asp)4 complex may be related to the scav-
have confirmed and extended the existing literature [8, enging of ÆO 2 and the prevention of
1
O2 daughter
9], and they are consistent with the SOD-mimetic reac- product formation in the skin or perhaps the induction
tivities of many small molecular mass copper complexes, of some antioxidant enzymes including Cu2Zn2SOD [8]
including Cu2(asp)4 and Cu(asp)2 complexes. Based following Cu(II) incorporation into apoSOD (Zn2SOD)
upon the direct ESR measurement of SOD-mimetic in vivo.
reactivity (Fig. 3), we have further confirmed that Cu(II) Because a number of ROS-generating systems such
compounds react with ÆO 2 directly when scavenging ÆO2
as XOD and NADPH oxidase are localized within cells
and that the Cu2(asp)4(DMSO)2 complex was the most and on cell membranes [38], membrane lipids are
active complex among the four different Cu(II) com- actually susceptible to ROS attack. In terms of pre-
pounds. venting lipid molecules from reactions with ROS,
Despite the relatively low SOD mimetic activity of Fisher et al [11] developed lipophilic Cu(II) complexes
Cu2(asp)4 and Cu(sal)2 compared to Cu2Zn2SOD, such that localize within lipid structures such as lipoproteins
compounds have been confirmed as enhancing cell sur- and cellular membranes. Measurement of the oil–water
vival following UVB irradiation (Figs. 4, 5). At present, partition coefficient (K=Coleyl alcohol/Cwater) of
it is difficult to unequivocally explain cell survival in the Cu2(asp)4 showed that Cu2(asp)4 has oil-soluble char-
presence of Cu(II) complexes following UVB irradia- acteristics, K=1.3, i.e., log P=0.1 [20]. This finding
tion; however, we speculate that Cu(II) compounds en- suggests that Cu2(asp)4 is a lipophilic complex, which
ter into cells and scavenge the generated ROS, or they can be transported to and localized in cell membranes
induce several Cu-dependent antioxidative proteins or and can scavenge ÆO 2 there.
enzymes such as Cu2Zn2SOD, which is known to en- Available Cu concentrations in both blood and skin
hance cell survival following UV and ionizing irradia- of mice treated orally on consecutive days with
tion injuries [10]. Cu2(asp)4 were found to be slightly increased, suggesting
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